CN202903792U

CN202903792U - ELISA (enzyme-linked immunoabsorbent assay) reagent kit for detecting ochratoxin A content

Info

Publication number: CN202903792U
Application number: CN 201220354004
Authority: CN
Inventors: 万宇平; 冯才伟; 陶光灿; 彭鸽; 吴小胜; 何丽霞; 张禹
Original assignee: Beijing Kwinbon Biotechnology Co Ltd
Current assignee: Beijing Kwinbon Biotechnology Co Ltd
Priority date: 2012-07-20
Filing date: 2012-07-20
Publication date: 2013-04-24
Anticipated expiration: 2022-07-20

Abstract

The utility model relates to an enzyme-linked immunoreagent kit for detecting ochratoxin A. The reagent kit comprises a 96-well enzyme plate, a concentrated enzyme conjugate, an enzyme conjugate diluent, a 6% standard solution, a substrate solution A, a substrate solution B, and a stop solution. By adopting a competitive ELISA method, a coupling antigen is pre-coated on enzyme plate microporous strips, the residual ochratoxin A in a sample competes with the coupling antigen pre-coated on the enzyme plate microporous strips for the enzyme conjugate of the ochratoxin A; a TMB (tetramethyl benzidine) substrate is used for color development; the absorbance value of the sample is in negative correlation with the content of the ochratoxin A which the sample contains; and the absorbance value of the sample is compared with a standard curve and multiplied by a corresponding dilution ratio to obtain the content of the ochratoxin A in the sample. Compared with an instrumental analysis technique, the enzyme-linked immunoreagent kit for detecting ochratoxin A has the characteristics of high sensitivity and the like, is convenient to use, and can play an important role in ochratoxin A residue detection.

Description

Detect the ELISA kit of ochratoxin A content

Technical field

The utility model relates to the kit of detection of drugs content, particularly detect enzyme linked immunosorbent detection (ELISA) kit of ochratoxin A content in the feed, ELISA(Enzyme Linked Immunosorbnent Assay) be the abbreviation of enzyme-linked immunosorbent assay, it is a kind of enzyme immune technology that grows up after immunofluorescence and radioimmunoassay technique.

Background technology

Ochratoxin A (Ochratoxin A) is produced by aspergillus and mould two class moulds, normal contaminated food products and feed.Ochratoxin A can cause the acute and chronic toxicity of animal, is strong carcinogen, can cause irreversible deadly murder by poisoning to kidney, cause the kidney atrophy, also can make fetal anomaly, miscarriage even death, have a strong impact on function and the upgrowth situation of animal, its potential hazard to the mankind receives much concern.For this reason, the JARC of international cancer research institution is located II category-B carcinogenic substance, and strengthening ochratoxin A is detected is to prevent from directly or indirectly being entered human foods chain important method by ochratoxin A contaminated food and feed.

At present, the residue analysis method of ochratoxin A mainly contains chemical analysis and immunochemical analyses method, chemical analysis is applied to the residue detection of toxin the earliest, namely become the official method that ochratoxin A detects in internationalization Epidemiological Analysis association (ACAC) thin layer chromatography in 1973, the advantage of thin layer chromatography is that method is simple, the reagent low price that uses, but exist sensitivity relatively poor, required reagent is various, and sense cycle is long, and instrument detects, have the characteristics such as sensitivity height such as liquid phase-MS, but the costliness of setting needs complicated sample pre-treatments, is not suitable for the examination of on-site supervision and great amount of samples.

The utility model content

Problem to be solved in the utility model provides a kind of ochratoxin A residue detection kit of small volume and less weight, it is easy and simple to handle, detect quick, accurate, highly sensitive, cost is low, good stability, can carry out simultaneously the detection of ochratoxin A content in a plurality of samples, reduce the needed time of detection sample.

The technical solution of the utility model is: a kind of ELISA kit that detects ochratoxin A content, comprise ELISA Plate, cover plate film, 11 bottles of reagent in 96 holes in box body and the box and put the recessed bottle position of reagent, a valve bag, it is characterized in that: ELISA Plate is to adopt 96 hole agent plate as solid phase carrier, the check-out console that pre-coated ochratoxin A coupled antigen is made on the kit capillary strip, 11 bottles of reagent are respectively 6 bottles of standard solutions, concentrated enzyme conjugates, enzyme combination diluent, substrate solution A liquid, substrate solution B liquid, stop buffer.

The kit box body is carton box; The polystyrene ELISA Plate in 96 holes is put in the vacuum aluminium foil bag; The cover plate film is plastic hard membrane; Standard solution is all used the white plastic bottle of white cap, concentrated enzyme conjugates is with the white PE plastic bottle of red cap, enzyme combination diluent is with the white PE plastic bottle of green cap, substrate solution A liquid is with the white PE plastic bottle of white cap, substrate solution B liquid is with the black PE plastic bottle of red cap, stop buffer is made by plastic foam with the white PE plastic bottle of yellow cap, recessed bottle position.

ELISA Plate is comprised of outer frame support and removable 12 enzyme marks reaction capillary strips of being placed on it, and each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes, the pre-coated ochratoxin A coupled antigen of each reacting hole; Cover plate film size and ELISA Plate square section in the same size; 6 bottles of standard solutions, the 1ml/ bottle; 1 bottle of concentrated enzyme conjugates, 0.7ml; 1 bottle of enzyme combination diluent, 14ml; 1 bottle of substrate solution A liquid, 7ml; 1 bottle of substrate solution B liquid, 7ml; 1 bottle of stop buffer, 7ml.

Reagent is with the plastic bottle splendid attire of different sizes, different colours and be fixed in the polyfoam mould and together be encapsulated in the carton box with ELISA Plate, cover plate film, valve bag, so that carry and transport.

Reagent in each kit enough carries out 96 times and measures (comprising the standard analysis hole), both can carry out the detection of a plurality of samples of one-time continuous, also can take plate hole apart repeated detection.Put back in the aluminium foil bag with the valve bag sealing accuracy that like this can guarantee reagent box testing result with remaining.During detection, ochratoxin A in the sample will be competed with pre-coated coupled antigen on the ELISA Plate capillary strip enzyme conjugates of ochratoxin A, develop the color with tmb substrate, the content of ochratoxin A becomes negative correlation in sample absorbance and the sample, multiply by more again its corresponding extension rate with typical curve, can draw ochratoxin A content in the sample, it is easy to operation.

Our experiments show that ochratoxin A is residual in this kit detection feed (raw material, batch and concentrate feed) sample, have very high sensitivity (1 μ g/L), the lowest detection of sample is limited to 10 μ g/kg, and the recovery is 90% ± 20%.The required instrument of this kit is less, only needs microplate reader, homogenizer, oscillator, hydro-extractor, micro sample adding appliance etc., and generally all there is outfit in similar laboratory, and required cost is lower.

The beneficial effects of the utility model are: can be used for quick, easy, exactly the detection of ochratoxin A content.

Description of drawings

Fig. 1 is the lateral longitudinal sectional view (the long 8.55cm of being) of the utility model ELISA Plate;

Fig. 2 is the side drawing in side sectional elevation (the long 12.8cm of being) of the utility model ELISA Plate;

Fig. 3 is the vertical view of the utility model ELISA Plate;

Fig. 4 is the utility model cover plate membrane plane figure;

Fig. 5 is the utility model reagent bottle longitudinal profile planimetric map;

Fig. 6 is the utility model fixed foam mould vertical view;

Fig. 7 is the utility model box body and fixed foam die side view.

Embodiment

Referring to accompanying drawing: enzyme mark reaction capillary strip (2) pre-coated ochratoxin A coupled antigens (3) are fixed on the outer frame support (1) of ELISA Plate, and enzyme mark reaction capillary strip (2) can be with requiring dismounting; Capping enzyme mark reaction capillary strip (2) when cover plate film (4) is put water-bath or constant temperature oven internal reaction for ELISA Plate, cover plate film size and ELISA Plate square section in the same size; The white plastic reagent bottle (5) of white cap is used for encapsulation 7ml substrate solution A liquid, the black plastic reagent bottle (5) of red cap is used for encapsulation 7ml substrate solution B liquid, the white plastic reagent bottle (5) of yellow cap is used for encapsulation 7ml stop buffer, the white plastic reagent bottle (6) of red cap is used for the concentrated enzyme conjugates of encapsulation 0.7ml, the white plastic reagent bottle (6) of green cap is used for encapsulation 14ml enzyme combination diluent, and the white plastic bottle (7) of white cap is used for the standard solution of encapsulation 1ml/ bottle; Polyfoam mould (8) bottle position placement location is followed successively by: 7ml substrate solution A liquid bottle position (10), 7ml substrate solution B liquid bottle position (11), 7ml stop buffer bottle position (12), 14ml enzyme combination diluent bottle position (13), 0.7ml concentrated enzyme conjugates bottle position (14), the standard solution bottle position (15) of 6 bottles of various concentration of 1ml/ bottle, box body (9) is carton box.

(1) use kit to detect the operation steps of ochratoxin A:

1, required reagent is taken out from cold storage environment, place room temperature (20-25 ℃/68-77 ℉) to rise again, note to shake up before every kind of liquid reagent uses.

2, take out the microwell plate that needs quantity, no microwell plate is put into valve bag, be stored in 2-8 ℃.

3, numbering: the corresponding micropore of sample and standard items is numbered according to the order of sequence, and it is parallel that each sample and standard items are done 2 holes, and the position at record standard hole and sample aperture place.

4, enzyme conjugates working fluid preparation: amount will concentrate enzyme conjugates and dilute (i.e. 20 parts of enzyme combination diluents of 1 part of concentrated enzyme conjugates adding) with enzyme combination diluent according to the volume of 1:20 as required.

5, add standard items/sample, enzyme conjugates working fluid: add standard items/sample 20 μ l in the micropore of correspondence, add immediately enzyme conjugates working fluid 100 μ l/ holes, the mixing that vibrates gently is with reacting 10min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate.

6, wash plate: carefully open the cover plate film, liquid in the hole is dried, fully wash 4-5 time with deionized water 250 μ l/ holes, every minor tick 10s pats dry with thieving paper.

7, colour developing: add substrate solution A liquid 50 μ l/ holes, add substrate solution B liquid 50 μ l/ holes again, the mixing that vibrates gently is with reacting 5min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate.

8, measure: add stop buffer 50 μ l/ holes, the vibration mixing is set microplate reader in the 450nm place, measures every hole OD value.

(2) result judges

(1) calculating of percentage absorptance, the percentage absorptance of standard items or sample equal the mean value (diplopore) of percentage absorbance of standard items or sample divided by the absorbance of first standard (0 standard), multiply by 100%, namely again

The mean light absorbency value of B-standard items or sample solution

The mean light absorbency value of B0-0 μ g/L standard solution

(2) drafting of typical curve and calculating

Take standard items percentage absorptance as ordinate, take the logarithm of ochratoxin A standard items concentration as horizontal ordinate, the drawing standard curve map.In the percentage absorptance substitution typical curve with sample, read the corresponding concentration of sample from typical curve, multiply by the actual concentrations that its corresponding extension rate is ochratoxin A in the sample.

Claims

1. ELISA kit that detects ochratoxin A content, comprise ELISA Plate, cover plate film, 11 bottles of reagent in 96 holes in box body and the box and put the recessed bottle position of reagent, a valve bag, it is characterized in that: ELISA Plate is to adopt 96 hole agent plate as solid phase carrier, the check-out console that pre-coated ochratoxin A coupled antigen is made on the kit capillary strip, 11 bottles of reagent are respectively 6 bottles of standard solutions, concentrated enzyme conjugates, enzyme combination diluent, substrate solution A liquid, substrate solution B liquid, stop buffer.

2. kit according to claim 1, it is characterized in that: box body is carton box; The polystyrene ELISA Plate in 96 holes is put in the vacuum aluminium foil bag; The cover plate film is plastic hard membrane; Standard solution is all used the white plastic bottle of white cap, concentrated enzyme conjugates is with the white PE plastic bottle of red cap, enzyme combination diluent is with the white PE plastic bottle of green cap, substrate solution A liquid is with the white PE plastic bottle of white cap, substrate solution B liquid is with the black PE plastic bottle of red cap, stop buffer is made by plastic foam with the white PE plastic bottle of yellow cap, recessed bottle position.

3. kit according to claim 1, it is characterized in that: ELISA Plate is comprised of outer frame support and removable 12 enzyme marks reaction capillary strips of being placed on it, each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes, the pre-coated ochratoxin A coupled antigen of each reacting hole; Cover plate film size and ELISA Plate square section in the same size; 6 bottles of standard solutions, the 1ml/ bottle; 1 bottle of concentrated enzyme conjugates, 0.7ml; 1 bottle of enzyme combination diluent, 14ml; 1 bottle of substrate solution A liquid, 7ml; 1 bottle of substrate solution B liquid, 7ml; 1 bottle of stop buffer, 7ml.